Study on Antioxidant Activity of Nuciferine of Lotus Leaf in Vitro

Abstract

Background: nuciferine was an aporphine alkaloid and one of the main components of lotus leaf. It has the effect of reducing blood lipids, anti-inflammatory and antioxidant. Objective: To study on antioxidant activity of nuciferine of lotus leaf in vitro. Ultrasonic extraction was devoted to extract nuciferine from lotus leaves. Methods: Chemical antioxidant method was used to investigate the scavenging ability of hydroxyl radical (·OH), superoxide anion radical (O^2.−), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS^•+) radical, and the ferric reducing ability of plasma (FRAP) of nuciferine. Cellular antioxidant assays were used to study the effects of nuciferine on lipid peroxidation and oxidative stress products (malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT)). Results: Chemical antioxidant experiments showed that lotus leaf extract (containing nuciferine about 67%) and nuciferine reference (98%) had good scavenging activity against ·OH, O^2.−, DPPH and ABTS^•+ radical, and had weak reducing ability to iron ions. Cellular antioxidant experiments illustrated that lotus leaf extract in low, medium and high dose could reduce lipid peroxidation by reducing MDA levels, and alleviate oxidative stress by increasing antioxidant enzyme activity (total GSH,SOD and CAT). Conclusion: The lotus leaf extract demonstrates remarkable in vitro antioxidant activity, exhibiting excellent scavenging effects on multiple free radicals, showing significant application potential in food and pharmaceutical fields.

Keywords

lotus leaf nuciferine antioxidant activity scavenges free radicals antioxidant enzyme activity

Introduction

Reactive oxygen species (ROS) were molecules or ions with reactive physical and chemical properties, including hydrogen peroxide (H₂O₂), hydroxyl radical (·OH) and superoxide anion radical (O^2.−), etc¹ Reactive oxygen species was involved in various biological processes within living organisms. However, excess ROS causes cell damage, tissue damage, and oxidative stress, which can lead to cancer, cardiovascular disease and neurological diseases.²

Antioxidants were substances that scavenge free radicals to prevent oxidative damage to cells and tissues or increasing the activity and number of antioxidant enzymes in organisms through a variety of pathways.³ Antioxidants were divided into inartificial (eg, natural plants)⁴ and synthetic (eg, Tert-butyl-p-cresol (BHT) and Metal chelators) antioxidants.⁵ However, natural antioxidants were safer compared to synthetic antioxidants.⁶ Now, antioxidants were increasingly being used to combat disease.⁷

Lotus leaf belongs to the Nelumbo nucifera Gaertn, which is rich in resources in the world and has extensive medicinal values. Nuciferine was an aporphine alkaloid and one of the main components of lotus leaf.⁸ It has the effect of reducing blood lipids,⁹ anti-inflammatory¹⁰ and antioxidant.¹¹ However, there has not been much research on the antioxidant capacity of nuciferine in vitro. Therefore, in this experiment, acid-assisted ultrasound¹² was applied to draw nuciferine from lotus leaf, and then the chemical antioxidant activity and cellular antioxidant activity of nuciferine were studied.

Chemical antioxidant method refers to the method in which the antioxidant capacity of a substance was manifested in scavenging free radicals, inhibiting pro-oxidants and reducing power, etc It was currently the main means to assess the antioxidant activity of substances, as well as it has the advantages of simplicity and speed.¹³ Cellular antioxidant method was an in vitro antioxidant method based on cell culture. This method was to explore how the target substance removes reactive oxygen species from cells under the induction of the environment or substance.¹⁴

Method

Reagents and Apparatus

Lotus leaf (Nelumbo nucifera Gaertn) were purchased from Lu’an Danbeier Biotechnology Co., Ltd, and the year of collection is 2020. Lotus leaf extract (containing nuciferine about 67%) was derived from “Optimizing Ultrasonic Extraction and Purification of nuciferine With Response Surface Method” (experimented, written, and published by the authors). The data for the purity characterization of lotus leaf extract were in Ref.¹²

Nuciferine reference (98%) was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd RAW264.7 cell (No. TCM13) were obtained from the Cell Bank of the Chinese Academy of Sciences. Additional reagents were placed in Supplementary Table 1 (Table S1). Preparation of all solutions was in Supplementary Table 2 (Table S2).

Quartz enzyme label plate was purchased from Shanghai Baiqian Biotechnology Co., Ltd Microplate reader (Molecular Devices iD5) was purchased from Annolen Biotechnology Co., Ltd The cell culture incubator was obtained from Shanghai Hengyi Scientific Instrument Co., Ltd The microscope was purchased from Olympus. Biosafety cabinet was purchased from BIOBASE. Cryogenic high-speed centrifuge was purchased from Eppendorf.

Chemical Antioxidant Method

Hydroxyl Radical (·OH) Scavenging Ability

The salicylic acid means can evaluate the scavenging ability of the target substance to ·OH free radical.¹³ Different concentrations of lotus leaf extract and nuciferine reference solution (0.05-5.0 mg/mL) were made, and 50 μL of this solution was placed into a 96-well plate. Next, 50 μL 9 mmol/L FeSO₄ (Iron(II) sulfate) solution was added, 50 μL 6 mmol/L H₂O₂ was added and left at normal temperature for 10 min, and 50 μL 9 mmol/L salicylic acid solution was kept at normal temperature for 30 min. The absorbance at 510 nm was surveyed with an enzyme-labeled instrument. L-ascorbic acid was applied as the control. Ultrapure water was used as the blank.

Based on clearance curve, the scavenging ability of lotus leaf extract to ·OH free radicals was analyzed. Each sample was measured in triplicate copies. The scavenging rate formula was as follows:

Scavenging rate (%) = \frac{(A 0 - A 1)}{A 0} \times 100 %

(1)

Among them, A₁ was the absorbance of the test sample, and A₀ was the absorbance of the blank. The clearance curve was made with the scavenging rate (%) as the ordinate and the mass concentration (mg/mL) as the abscissa.

Superoxide Anion Radical (O^2.−) Scavenging Ability

Pyrogallol method was used to determine the scavenging ability of the target substance to O^2.− free radical.^15-17 Different concentrations of lotus leaf extract and nuciferine reference solution (0.05-5.0 mg/mL) were made, and 50 μL of this solution was placed into a 96-well plate. Then, 135 μL 50 mmol/L Tris-HCl (pH 8.2) buffer solution was added and reacted at 37 °C for 15 min. Next, 15 μL of 25 mmol/L pyrogallol solution was added, and the reaction was terminated with 50 μL of 8 mol/L HCl after 4 min at normal temperature. After standing for 30 min, the absorbance at 299 nm was surveyed with an enzyme-labeled instrument. The control and blank were the same as in “Hydroxyl radical (·OH) scavenging ability”.¹⁸

The calculation and clearance curve were the same as “Hydroxyl radical (·OH) scavenging ability”.

DPPH Radical Scavenging Ability

The DPPH way can estimate the antioxidant activity of substances in vitro.¹⁹ Different concentrations of lotus leaf extract and nuciferine reference solution (0.05-5.0 mg/mL) were made, and 50 μL of this solution was placed into a 96-well plate. Then, 0.05 mg/mL DPPH solution was put in, mix well and let stand at normal temperature for 15 min. The Absorbance at 517 nm was determined with an enzyme-labeled instrument. L-ascorbic acid was applied as the control. Methanol added to 0.05 mg/mL DPPH solution was blank 1, and methanol plus ethanol was blank 2.

According to the clearance curve, the scavenging ability of lotus leaf extract on DPPH radical was analyzed. Each sample was measured in triplicate copies. The scavenging rate formula was as follows:

Scavenging rate (%) = [1 - \frac{(A i - A j)}{(A 0 - A j)}] \times 100 %

(2)

Among them, A_i was the absorbance of the test sample, A₀ was the absorbance of the blank 1, and A_j was the absorbance of the blank 2. The ordinate of the clearance curve was the scavenging rate (%) and the abscissa was the mass concentration (mg/mL).

ABTS^•+ Radical Scavenging Ability

The ABTS method can monitor the antioxidant capacity of a substance.¹⁹ Different concentrations of lotus leaf extract and nuciferine reference solution (0.05-5.0 mg/mL) were made, and 50 μL of this solution was placed into a 96-well plate. Then, ABTS•+ and K₂S₂O₈ (Potassium persulfate) mixtures was added, mixed well and left to stand for 15 min at normal temperature. Absorbance at 734 nm was determined with an enzyme-labeled instrument. L-ascorbic acid was applied as the control. Methanol added to ABTS^•+ and K₂S₂O₈ mixtures was blank 1, and methanol plus purified water was blank 2.

The calculation and clearance curve were the same as “DPPH radical scavenging ability”.

Determination of Ferric Reducing Ability of Plasma (FRAP)

FRAP was capable of analyzing the iron ion reduction capacity of a substance.¹⁹ Different concentrations of lotus leaf extract and nuciferine reference solution (0.05-5.0 mg/mL) were made, and 20 μL of this solution was placed into a 96-well plate. Next, 200 μL FRAP mixtures was added, mixed well, and let stand at 37 °C for 20 min. The absorbance was determined with an enzyme-labeled instrument at 593 nm. BHT was the control. Methanol was blank.

According to FRAP curve, the reducing ability of lotus leaf extract was analyzed. Each sample was measured in triplicate copies. FeSO₄ equivalent (FRAP value) was calculated based on a FeSO₄ standard curve. The formula for FRAP value was as follows:

FRAP value = \frac{(A i - A j) - b}{k}

(3)

Among them, A_i was the absorbance of the test sample, A_j was the absorbance of the blank. b and k were the intercepts and slopes of the FeSO₄ standard curve, respectively. The unit of FRAP value was mmol/L/g.

The FeSO₄ standard curve of concentration-absorbance was drew with the concentration (0.05-1.2 mmoL/L) and (A_i − A_j) of FeSO₄ as the horizontal and vertical coordinates, respectively.

Cellular Antioxidant Method

Cell culture.²⁰

RAW264.7 cells were fostered with DMEM (adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin bispecific antibody) under conditions of 37 °C and 5% CO₂. During cell culture, the cells were fed once every 24 h and passaged once every 48 h, and the experiment was carried out with the cells of logarithmic growth stage.

Establishment of Cell Damage Models Using H₂O₂

Based on relevant literature reports, an H₂O₂-induced cell injury model was established.²⁰ RAW264.7 cells were inoculated on a 96-well plate at a density of 2.0 × 10⁵/mL and cultured under 37 °C and 5% CO₂ for 24 h. The media were replaced with fresh DMEM including different concentrations of H₂O₂ (0-2000 μmol/L) (dissolved in DMSO) and incubation for 24 h. Then, the supernatant was discarded, 100 μL CCK8 solution was added and cultured for 40 min. Next, the cell culture was stopped, and the supernatant was removed to determine the absorbance at 450 nm. Each concentration was provided with 3 multiple holes. Cell viability was analyzed based on Formula 4 as follows:

Cell viability (%) = (1 - \frac{A 0 - A 1}{A 0}) \times 100

(4)

Among, A₀ was the blank and A₁ was the sample. The curve was plotted in terms of concentration as the abscissa and the ordinate of cell viability (%).

Concentration Screening of Lotus Leaf Extract

The experimental process of screening was the same as “Establishment of cell damage models using H₂O₂”, and H₂O₂ was replaced with different concentrations of lotus leaf extract (1-5000 μg/mL).

Determination of MDA, Total GSH, SOD, and CAT Levels in Cells

Antioxidant enzymes were quantified with reference to relevant literature.^20-22 2 mL RAW264.7 cells with density of 2.0 × 10⁵/mL were added to each well of the 6-well plate and cultivated at 37 °C and 5% CO₂ for 24 h. After discarding the culture medium, tests were carried out according to Table 1.

Table 1.

Experiment Operations.

Test	Operation
Blank	Add 2 mL DMEM solution, discard the supernatant after 24 h, add 2 mL DMEM solution, and isolate proteins after 4 h
Model	Add 2 mL DMEM solution, discard the supernatant after 24 h, add 2 mL H₂O₂ culture medium (600 μmol/L), and isolate proteins after 4 h
Control	Add 2 mL L-ascorbic acid culture medium (150 μg/mL), discard the supernatant after 24 h, add 2 mL H₂O₂ culture medium (600 μmol/L), and isolate proteins after 4 h
Low-dose	Add 2 mL lotus leaf extract culture medium (50 μg/mL), discard the supernatant after 24 h, add 2 mL H₂O₂ culture medium (600 μmol/L), and isolate proteins after 4 h
Medium-dose	Add 2 mL lotus leaf extract culture medium (100 μg/mL), discard the supernatant after 24 h, add 2 mL H₂O₂ culture medium (600 μmol/L), and isolate proteins after 4 h
High-dose	Add 2 mL lotus leaf extract culture medium (150 μg/mL ”), discard the supernatant after 24 h, add 2 mL H₂O₂ culture medium (600 μmol/L),and isolate proteins after 4 h

Protein isolation refers to the isolation of proteins from experimental cells. The cells treated in Table 1 were placed in an ice bath and immediately added 250 μL RIPA lysis buffer for 10 min to completely lysate the cells. The fully lysed cells were then placed in a 4 °C centrifuge at a speed of 12 000 r/min for 15 min to obtain the isolated proteins. The protein was quantified by BCA protein quantitation kit. Activity of MDA, total GSH, SOD and CAT was determined according to the method of the corresponding kit.

Statistical Methods

ANOVA was used for between-group differences. ANOVA for all cell data was provided in Supplementary Table 3 (Table S3). The average ± SEM of the three independent experiments was applied to represent the experimental data. The P-value < .05 considered to be a significant difference. OriginPro version 2024 and SPSS version 27 were applied for drawing and statistical analysis, respectively.

Results

Chemical Antioxidant Activity

Hydroxyl Radical (·OH) Scavenging Activity

The results of scavenging ability of lotus leaf extract, nuciferine reference and l-ascorbic acid to ·OH radical were shown in Figure 1A. As the concentrations of lotus leaf extract, nuciferine reference and l-ascorbic acid increase, their scavenging activity gradually fortifies until a plateau was reached. The scavenging activity of lotus leaf extract and nuciferine reference to ·OH radicals was above l-ascorbic acid in the range of 0-1.0 mg/mL, but below it in the range of 1.5-5.0 mg/mL. This illustrates that lotus leaf extract and nuciferine reference have good ·OH radical scavenging capacity in the range of 0-5.0 mg/mL.

Figure 1.

Chemical Antioxidant Activity. (A) Scavenging Activity of ·OH Radical. (B) Scavenging Activity of O^2.− Radical. (C) Scavenging Activity of DPPH Radical. (D) Scavenging Activity of ABTS^•+ Radical. (E) FeSO₄ Standard Curve (0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.20 mmol/L). (F) Ferric Reducing Ability of Plasma. The Concentration of lotus Leaf Extract, Nuciferine Referencer, l-Ascorbic Acid and BHT was all 0.05-5.0 mg/mL (0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0 mg/mL). The Average ± SEM of the Three Independent Experiments was Used to Represent the Experimental Data.

Superoxide Anion Radical (O^2.−) Scavenging Activity

As shown in Figure 1B, the scavenging capacity of lotus leaf extract and nuciferine reference on O^2.− radical was lower than that of l-ascorbic acid. With the increase of the concentrations of lotus leaf extract, nuciferine referencer and l-ascorbic acid, their scavenging rate rised until they reached a plateau. This illustrates that the O^2.− radical scavenging ability of lotus leaf extract and nuciferine referencer was better, and it was close to l-ascorbic acid.

DPPH Radical Scavenging Activity

In Figure 1C, the DPPH radical scavenging capacity of l-ascorbic acid was better than that of lotus leaf extract and nuciferine referencer. Scavenging activity of lotus leaf extract, nuciferine referencer and l-ascorbic acid against DPPH radical augments with increasing concentration. Their scavenging activity was almost equivalent at a concentration of 5 mg/mL. The above results indicate that lotus leaf extract and nuciferine referencer have good DPPH radical scavenging ability.

ABTS^•+ Radical Scavenging Activity

From Figure 1D, we can obtain results that the scavenging ability of lotus leaf extract and nuciferine referencer on ABTS^•+ radical was weaker than that of l-ascorbic acid. With the increase of concentration, the scavenging rate of lotus leaf extract, nuciferine referencer and l-ascorbic acid rises until they reach a plateau of 2.5 mg/mL, at which they had almost the same scavenging activity on ABTS^•+ radical. This illustrates that the ABTS^•+ radical clearance capacity of lotus leaf extract and nuciferine referencer was better and close to l-ascorbic acid.

Ferric Reducing Ability of Plasma (FRAP)

Different concentrations of FeSO₄ solution were configured. The standard curve obtained was shown in Figure 1E. The linear equation was y = 0. 66116x − 0.00173 and the correlation coefficient was 0.9997 (R = 0.9997).

As shown in Figure 1F, the FRAP value of lotus leaf extract and nuciferine referencer was significantly lower than that of BHT. With the augment of concentration, the FRAP value in lotus leaf extract and nuciferine referencer hardly increases, but BHT augments substantially. This indicates that the FRAP of lotus leaf extract and nuciferine referencer was weak.

Cellular Antioxidant Activity

600 μmol/L H₂O₂ as the Modeling Concentration of Cell Damage

H₂O₂ can induce oxidative stress in cells, so it was used to construct oxidative damage models in RAW264.7 cells. In Figure 2A, cell viability gradually decreases as the concentration of H₂O₂ increases. Different concentrations of H₂O₂ inhibited cell activity. If the concentration of H₂O₂ was too high, it will cause a large number of cell death, and if it is too low, the cell damage will not be obvious. Therefore, the concentration of H₂O₂, which has a cell viability of about 50%, was selected for the construction of the oxidative damage model. In this experiment, 600 μmol/L H₂O₂ (cell activity 57.65%) was selected as the modeling concentration.

Figure 2.

Effects of H₂O₂ and Lotus Leaf Extract on the Activity of RAW264.7 Cells. (A) The Effect of Different Concentrations of H₂O₂ (0, 200, 400, 600, 800, 1000, 1200 μmol/L) on the Activity of RAW264.7 cells. (B) The Effect of Different Concentrations of Lotus Leaf Extract (1, 10, 25, 50, 100, 150, 200, 500, 1000, 2000, 5000 μg/mL) on the Activity of RAW264.7 Cells. The Average ± SEM of the Three Independent Experiments was Used to Represent the Experimental Data.

Screening Results of Concentration of lotus Leaf Extract

Figure 2B shows that cell viability first increases and then decreases as the concentration of lotus leaf extract rises. The concentration of lotus leaf extract at 1-200 μg/mL promotes cell growth and inhibits cell growth at 200-5000 μg/mL. Therefore, concentrations that significantly promoted cell growth were selected for the low-, medium- and high-dose. The concentrations in the low-, medium- and high-dose were 50, 100, and 150 μg/mL, respectively. Due to the two-way regulatory effect of lotus leaf extract, its EC₅₀ was 78.4 μg/mL (95%CI 65.2-94.1) and its IC₅₀ was 1240 μg/mL (95%CI 980-1570) (n = 3).

Evaluation of Lipid Peroxidation and Antioxidant Enzyme Activity of lotus Leaf Extract

To study the antioxidant activity of lotus leaf extract in vitro, lipid peroxidation and antioxidant enzyme activities of cells treated with lotus leaf extract (containing nuciferine about 67%) were evaluated under oxidative stress, as shown in Figure 3.

Figure 3.

Evaluation of Lipid Peroxidation and Antioxidant Enzyme Activity of lotus Leaf Extract. (A) The Effect of lotus Leaf Extract on the Cellular Concentration of MDA. (B) The Effect of lotus Leaf Extract on the Activity of Total GSH. (C) The Effect of lotus Leaf Extract on the Activity of SOD. (D) The Effect of lotus Leaf Extract on the Activity of CAT. The Average ± SEM of the Three Independent Experiments was Used to Represent the Experimental Data. *P < .001, ANOVA Analyses.

In Figure 3A, there was a significant difference in MDA levels between the blank and model, suggesting that ROS caused lipid peroxidation to occur dramatically (P < .001). In the low-, medium- and high-dose, lotus leaf extract can significantly reduce the level of MDA (P < .001). Among them, the middle-dose had the best performance in reducing MAD. Compared with the control, the ability of lotus leaf extract to reduce MDA was weaker than that of l-ascorbic acid (P < .001). This illustrates that lotus leaf extract had better lipid antioxidant capacity, but lower than l-ascorbic acid.

Figure 3B shows that there was a significant difference in total GSH content between the blank and the model, indicating that ROS inhibits the production of antioxidant enzymes (P < .001). The low-, medium-, and high-dose recovered the levels of total GSH to varying degrees, with the most pronounced recovery of total GSH levels in the middle-dose (P < .001). The ability of lotus leaf extract to increase total GSH was almost comparable to that of l-ascorbic acid compared to the control (P < .001).

As shown in Figure 3C, there was a significant difference in SOD levels between the blank and model (P < .001). In the low-, medium- and high-dose, SOD levels recovered significantly (P < .001). The recovery effect was most pronounced in the medium-dose and was comparable to that of the control.

As we can see from Figure 3D, there was a significant difference in CAT content between the blank and model. Activity of CAT was significantly increased in the low-, medium- and high-dose. Among them, CAT activity was almost identical in the medium- and high-dose. The CAT level in the dose was slightly lower than in the control. The above results suggest that lotus leaf extract protects cells from ROS damage by augmenting the activity of antioxidant enzymes (total GSH, SOD, and CAT).

Discussion

The antioxidant activity of lotus leaf extract (containing nuciferine about 67%) in vitro was determined by various measurements.

·OH radical was reactive oxygen species that interact with salicylic acid to produce a substance with strong UV absorption at 510 nm. When antioxidant was present, this colored substance decreases due to the disappearance of ·OH radical.¹³ In this paper, the scavenging activity of lotus leaf extract and nuciferine reference against ·OH radical was significant, and the scavenging activity fortified with the increase of concentration in the range of 0.05-5.0 mg/mL. O^2.− radical was also a reactive oxygen species. Pyrogallone produces O^2.− radical under weakly alkaline conditions, which promotes the formation of a substance with strong UV absorption at 299 nm. When antioxidant was added, it hinders the formation of this colored substance.¹⁵ In this study, lotus leaf extract and nuciferine reference had a better clearance of O^2.− radical and were close to l-ascorbic acid. The DPPH means was often applied to analyze the antioxidant activity of a substance. There were lone electron pairs in DPPH radical, and there was strong UV absorption at 517 nm. When an antioxidant was present, it binds to lone electron pairs to attenuating UV absorption.¹⁹ In this experiment, lotus leaf extract and nuciferine reference had good DPPH radical scavenging capacity, and the scavenging activity was close to that of l-ascorbic acid. The ABTS^•+ means was applied to analyze the antioxidant activity of substances. The ABTS^•+ radical have strong UV absorption at 734 nm. As antioxidant were added, their interaction with ABTS^•+ weakens UV absorption.¹⁹ In this study, lotus leaf extract and nuciferine reference were highly effective in scavenging ABTS^•+ radical, and had almost the same scavenging capacity as l-ascorbic acid in the range of 2.0-5.0 mg/mL. The FRAP way can be used to analyze the reducing capacity of antioxidant. This method was based on Fe²⁺ and Tripyridyltriazine (TPTZ) to produce colored substances with UV absorption at 593 nm. Antioxidant can reduce these colored substances to reduce UV absorption.¹⁹ In this experiment, the reducing ability of lotus leaf extract and nuciferine reference was weaker and significantly weaker than that of BHT.

Alkaloids in lotus leaves have been shown to protect against ethanol-induced liver injury by scavenging ROS.^23,24 In this experiment, lotus leaf extract in the low and medium dose could clear ROS, but the scavenging effect of ROS in the high dose was weaker than that in the medium dose. MDA was a lipid peroxidation product that can reflect the degree of lipid peroxidation.²⁵ The level of MDA can be significantly decreased in low, medium and high dose. GSH removes O^2.− radical and H₂O₂ to reduce oxidative stress.²⁶ SOD was capable of converting O^2.− radical to molecular oxygen (O₂) or H₂O_2.²⁷ CAT can dissociate H₂O₂ into molecular oxygen (O₂) and water.²⁸ In this paper, antioxidant enzyme (total GSH, SOD, and CAT) activity can be significantly elevated in the low-, medium-, and high-dose. Among them, the middle-dose had the strongest antioxidant capacity compared with the low- and high-dose. In this paper, we report a dose-dependent stimulation effect at low concentrations (1-200 μg/mL) (EC₅₀ = 78.4 μg/mL) and inhibition at high concentrations (>200 μg/mL) (IC₅₀ = 1240 μg/mL). This bidirectional regulatory pattern suggests that lotus leaf extract may affect target cell activity through different mechanisms. However, the current calculation of the IC₅₀ is based on a four-parameter model of the inhibition phase, and the bidirectional effect may lead to the IC₅₀ not being fully applicable. Therefore, future studies may need to detect cell cycle arrest at 200 μg/mL turning points and expand the concentration gradient (50-500 μg/mL) to improve accuracy.

This study evaluated the in vitro antioxidant capacities of various lotus leaf extracts (Table 2). Compared with previous reports, our investigation employed a more comprehensive analytical approach, including multiple free radical scavenging activities (DPPH, ABTS, hydroxyl, and superoxide anion), ferric reducing antioxidant power (FRAP), various antioxidant enzyme activities (SOD, CAT, toral GSH), and oxidative stress biomarkers (MDA). It can be found in Table 2, Nymphaea lotus L. exhibit high DPPH free radical scavenging capacity (IC₅₀ = 0.0976 mg/mL). The Nelumbo nucifera extracts in our study demonstrated superior antioxidant activities compared to most literature values, exhibiting exceptional ABTS radical scavenging capacity (99.33% at 2 mg/mL) and remarkable superoxide anion (O^2.−) scavenging efficiency (83.83%). Moreover, flavonoid-rich extracts (particularly those containing rutin) consistently exhibited enhanced free radical scavenging capacity. The reduction in MDA levels (1.9 μmol/mg) coupled with elevated GSH (65.53 μmol/mg) and SOD activity (96.04 U/mg) suggests that nuciferine effectively mitigates oxidative cell damage. In this study, nuciferine demonstrated broad-spectrum antioxidant properties. However, certain limitations should be noted: (1) some literature failed to provide precise concentration data; (2) methodological variations existed across studies, particularly in the quantification approaches (eg, scavenging rate%, and IC₅₀ values);(3) Since lotus leaf extracts contain diverse components, direct comparisons between them have limitations.

Table 2.

Comparison of the Antioxidant Capacity of Different lotus Leaf Extracts.

Plant	Species	Substance	Main Components	Extract Concentration	Antioxidant Capacity
Lotus leaf²⁹	Nelumbo nucifera Gaertn	DPPH radical	Gallic acid, rutin	60 μg/mL	scavenging rate = 91.23%
		ABTS^•+ radical		200 μg/mL	scavenging rate = 95.09%
		Ferrous Ions (Fe²⁺)		200 μg/mL	FRAP = 0.67
Lotus leaf³⁰	Zizyphus lotus L.	DPPH radical	Total phenols, total flavonoids	/	IC₅₀ = 2.896 ± 0.85 μg/mL
Lotus leaf³⁰	Zizyphus lotus L.	Ferrous Ions (Fe²⁺)	Total phenols, total flavonoids	/	EC₅₀ = 99.435 ± 0.72 μg/mL
Lotus leaf³¹	Nelumbo nucifera Gaertn	DPPH radical	Liriodenine, lysicamine, Anonaine, ect.	100 μM	scavenging rate = 35.6%
Lotus leaf³¹	Nelumbo nucifera Gaertn	ABTS^•+ radical	Liriodenine, lysicamine, Anonaine, ect.	100 μM	scavenging rate = 40.2%
Lotus leaf³²	Nelumbo nucifera Gaertn	SOD	flavonoid	200 μg/mL	80 U/mL
		CAT		200 μg/mL	30 U/mL
		GSH		200 μg/mL	3 mg GSH/L
		GSH-Px		200 μg/mL	800 U/mL
		MDA		200 μg/mL	2 nmol/mL
Lotus leaf³³	Nymphaea lotus L.	DPPH radical	Total phenols, total flavonoids	/	IC₅₀ = 0.0976 ± 0.25 mg/mL
		Lipid peroxidation		/	IC₅₀ = 0.2367 ± 0.006 mg/mL
		Ferrous Ions (Fe²⁺)		/	IC₅₀ = 0.447 ± 0.02 mg/mL
		Ferrous Ions (Fe³⁺)		/	IC₅₀ = 0.151 ± 0.03 mg/mL
Lotus leaf³⁴	Nelumbo nucifera Gaertn	GSH	Total flavonoids, gallic acid and rutin	100 μg/mL	7.5 mgGSH/g
		MDA		500 mg/kg	3.54 ± 1.205 nmoL/mg
		GSH-Px		500 mg/kg	48.41 ± 3.25 U/mg
		CAT		500 mg/kg	3.95 ± 0.58 U/mg
		SOD		500 mg/kg	51.18 ± 4.52 U/mg
Lotus leaf (this work)	Nelumbo nucifera Gaertn	hydroxyl radical (·OH)	nuciferine	5.0 mg/mL	scavenging rate = 50.77 ± 0.084%
		superoxide anion radical (O^2.−)		5.0 mg/mL	scavenging rate = 83.83 ± 0.159%
		DPPH radical		5.0 mg/mL	scavenging rate = 92.32 ± 0.182%
		ABTS^•+ radical		2.0 mg/mL	scavenging rate = 99.33 ± 0.078%
		Ferrous Ions (Fe²⁺)		5.0 mg/mL	FRAP = 0.17 ± 0.00022
		MDA		100 μg/mL	1.9 ± 0.047 μmol/mg
		Total GSH		100 μg/mL	65.53 ± 0.687 μmol/mg
		SOD		100 μg/mL	96.04 ± 0.560 U/mg
		CAT		100 μg/mL	1.74 ± 0.009 U/mg

The antioxidant capacity of lotus leaf extract was compared with other plants, and the results are shown in Table 3. Compared with the other plants in Table 3, there was a lack of reference substance in this paper. We used only l-ascorbic acid and BHT as control experiments. However, the effects of the extract on the activity of various free radicals and antioxidant enzymes are studied in detail in this paper. Although lotus leaf extract did not exhibit better antioxidant capacity than the control, its non-toxic and harmless properties were also advantages. In the world, lotus leaf resources are extensive, and it can become a potential object of industrial production.

Table 3.

Comparison of the Antioxidant Capacity of Different Plants.

Plant	Species	Substance	Extract Concentration	Antioxidant Capacity
Marshmallow Flower (EEM)³⁵	Althaea officinalis L.	Total antioxidant activity	100 μg/mL	BHT > BHA > EEM > a-tocopherol
		DPPH radical		a-tocopherol > BHA > EEM > BHT
		Ferrous Ions (Fe²⁺)		EEM > BHA > a-tocopherol > BHT
		Superoxide Radical Anion		BHA > EEM > a-tocopherol > BHT
Allium species³⁶	Liliaceae	Total antioxidant activity	30 μg/mL	compound 3 > compound 1 > Trolox = compound 2 > crude extract > a-tocopherol
		Ferric ion (Fe³⁺)		BHT > crude extract > α-tocopherol > compound 3 > compound 1 = compound 2
		Ferrous Ions (Fe²⁺)		EDTA > crude extract > compound 2 > compound 3 > compound 1
		DPPH radical		crude extract > BHA > BHT>α-tocopherol > compound 2 > compound 3 > compound 1
Echinacea³⁷	Echinacea purpurea (L.) Moench and Echinacea pallida (Nutt.) Nutt	DPPH radical	IC₅₀	EPU-Flowers (MeOH)>EPU-leaves (MeOH)> EPA-leaves (MeOH)>EPA-Flowers (MeOH)> BHT > trolox > BHA
		ABTS^•+ radical	IC₅₀	EPU-Flowers (MeOH)>EPA-leaves (MeOH)> trolox > BHT > BHA
		Ferrous Ions (Fe²⁺)	40 μg/mL	BHA > EPA-leaves (H₂O)>EPA-Flowers(H₂O)>BHT > EPU-leaves (H₂O)=trolox
Lotus leaf (this work)	Nelumbo nucifera Gaertn	hydroxyl radical (·OH)	5 mg/mL	LA > nuciferine referrence > lotus leaf extract
		superoxide anion radical (O^2.−)		LA = nuciferine referrence > lotus leaf extract
		DPPH radical		LA = nuciferine referrence = lotus leaf extract
		ABTS^•+ radical		LA = nuciferine referrence = lotus leaf extract
		Ferrous Ions (Fe²⁺)		BHT > nuciferine referrence > lotus leaf extract
		MDA	100 μg/mL	LA < lotus leaf extract
		GSH		LA > lotus leaf extract
		SOD		LA = lotus leaf extract
		CAT		LA = lotus leaf extract

Lotus leaves are abundant in Asia, providing a foundation for industrial applications. Based on this study on the in vitro antioxidant activity of lotus leaf extract, it can be applied in fields such as food, cosmetics, and pharmaceuticals. As a natural antioxidant, it can serve as a functional food additive to extend the shelf life of food products, as well as be incorporated into cosmetics to scavenge free radicals and prolong cell longevity. Additionally, it may also act as a potential antioxidant to extend the shelf life of pharmaceuticals.

The limitations of this article are that it does not investigate the effects of different concentrations of nuciferine and lotus leaf extracts on free radical scavenging capacity and cellular antioxidant capacity. The sample size of this study was insufficient. This study only studied ·OH and O^2.− radical, and did not fully consider the effects on other oxygen free radicals (such as hydrogen peroxide, singlet oxygen, and peroxide lipids, etc). Furthermore, the article does not analyze the specific components contained in the lotus leaf extract, and only nuciferine was quantified by high performance liquid chromatography. These will be the focus of subsequent research.

Conclusion

This study systematically evaluated the in vitro antioxidant activity of lotus leaf extract containing 67% nuciferine. The extract demonstrated significant scavenging capacity against hydroxyl radicals (·OH), superoxide anions (O²⁻·), DPPH, and ABTS⁺· radicals, with scavenging rates ranging from 50.77% to 99.33% at concentrations of 2.0-5.0 mg/mL, comparable to L-ascorbic acid. The medium-dose group (100 μg/mL) exhibited the strongest overall antioxidant effects, significantly reducing malondialdehyde (MDA) levels to 1.9 μmol/mg while increasing glutathione (GSH) levels to 65.53 μmol/mg and enhancing superoxide dismutase (SOD) and catalase (CAT) activities to 96.04 U/mg and 1.74 U/mg, respectively. A bidirectional dose-response relationship was observed, where low concentrations (1.0-200 μg/mL, EC₅₀ = 78.4 μg/mL) stimulated antioxidant enzyme activity, while higher concentrations (>200 μg/mL, IC₅₀ = 1240 μg/mL) exhibited inhibitory effects. Compared to previous studies, the extract showed superior ABTS⁺· radical scavenging (99.33%) and superoxide anion elimination (83.83%). However, limitations include the lack of investigation into other reactive oxygen species (eg, hydrogen peroxide) and incomplete characterization of bioactive components beyond nuciferine. These findings highlight the potential of lotus leaf extract as a natural antioxidant for applications in food preservation, cosmetics, and pharmaceuticals. Future research should focus on elucidating the synergistic effects of its bioactive compounds and validating its efficacy in vivo.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251349923 - Supplemental material for Study on Antioxidant Activity of Nuciferine of Lotus Leaf in Vitro

Supplemental material, sj-docx-1-npx-10.1177_1934578X251349923 for Study on Antioxidant Activity of Nuciferine of Lotus Leaf in Vitro by Mengdie Wu, Tao Han, Hongmin Gao, Yang Zhou, Huan Zhu and Hongzhi Pan in Natural Product Communications

Footnotes

Acknowledgements

We gratefully acknowledge the financial support from the National Natural Science Fund of China (No.82273687). We also extend our sincere appreciation to Shanghai University of Traditional Chinese Medicine and Shanghai University of Medicine & Health Sciences for their academic nurturing. Special thanks to Professor Hongzhi Pan for his invaluable guidance.

ORCID iDs

Mengdie Wu

Tao Han

Hongmin Gao

Yang Zhou

Huan Zhu

Hongzhi Pan

Author Contributions

Mengdie Wu was in charge of experiments, collecting data and writing manuscripts. Tao Han was responsible for drawing, data analysis and reviewing the manuscript. Hongmin Gao, Yang Zhou and Huan Zhu were responsible for data analysis and investigation. Hongzhi Pan was responsible for reviewing, supervising, managing projects, and providing funding. All authors support the publication of the article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by National Natural Science Fund of China (No.82273687).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

All supporting data and results are included in this article and .

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

Please find the following supplemental material available below.

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Study on Antioxidant Activity of Nuciferine of Lotus Leaf in Vitro

Abstract

Keywords

Introduction

Method

Reagents and Apparatus

Chemical Antioxidant Method

Hydroxyl Radical (·OH) Scavenging Ability

Superoxide Anion Radical (O2.−) Scavenging Ability

DPPH Radical Scavenging Ability

ABTS•+ Radical Scavenging Ability

Determination of Ferric Reducing Ability of Plasma (FRAP)

Cellular Antioxidant Method

Cell culture. 20

Establishment of Cell Damage Models Using H2O2

Concentration Screening of Lotus Leaf Extract

Determination of MDA, Total GSH, SOD, and CAT Levels in Cells

Statistical Methods

Results

Chemical Antioxidant Activity

Hydroxyl Radical (·OH) Scavenging Activity

Superoxide Anion Radical (O2.−) Scavenging Activity

DPPH Radical Scavenging Activity

ABTS•+ Radical Scavenging Activity

Ferric Reducing Ability of Plasma (FRAP)

Cellular Antioxidant Activity

600 μmol/L H2O2 as the Modeling Concentration of Cell Damage

Screening Results of Concentration of lotus Leaf Extract

Evaluation of Lipid Peroxidation and Antioxidant Enzyme Activity of lotus Leaf Extract

Discussion

Conclusion

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251349923 - Supplemental material for Study on Antioxidant Activity of Nuciferine of Lotus Leaf in Vitro

Footnotes

Acknowledgements

ORCID iDs

Author Contributions

Funding

Declaration of Conflicting Interests

Data Availability Statement

Statement of Human and Animal Rights

Statement of Informed Consent

Supplemental Material

References

Supplementary Material

Superoxide Anion Radical (O^2.−) Scavenging Ability

ABTS^•+ Radical Scavenging Ability

Cell culture.²⁰

Establishment of Cell Damage Models Using H₂O₂

Superoxide Anion Radical (O^2.−) Scavenging Activity

ABTS^•+ Radical Scavenging Activity

600 μmol/L H₂O₂ as the Modeling Concentration of Cell Damage